CN103497892A - Cell culture substrate, and preparation method and application thereof - Google Patents
Cell culture substrate, and preparation method and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of biological material of tissue engineering. The invention discloses a cell culture substrate, a preparation method thereof, and an application thereof. According to the invention, umbilical cord fibroblast cell self-secretion substrate is adopted; and through decellularization treatment and drying and sterilization, a natural tissue engineering material is obtained. The material comprises collagen type I, collagen type III, fibronectin, laminin, decorin, and the like, and can be used in in-vitro amplification of target mesenchymal stem cells. With the cell culture substrate provided by the invention, mesenchymal stem cell in-vitro amplification efficiency can be substantially improved, special immune phenotype of stem cells can be maintained, and stem cell active oxygen content can be substantially reduced. Also, raw material source is wide, and no ethic and moral problem is caused.
Description
Technical field
The invention belongs to the technical field of biological materials of organizational project, be specifically related to a kind of cell cultures base material and its preparation method and application.
Background technology
Mescenchymal stem cell, due to its characteristic with height self-renewal capacity and multi-lineage potential, has been widely used in the fields such as regenerative medicine, gene therapy, organizational project, cytokine alternative medicine.
Although it is clinical that the mescenchymal stem cell treatment is marched toward just steadily, the mesenchymal stem cell transplantation technology is widely used in treating to still have facing many difficulties.The first, the quantity of transplanting mescenchymal stem cell: clinical effective transplanting requires mescenchymal stem cell to reach certain degree, but facts have proved and externally be difficult to obtain enough tissue-specific mescenchymal stem cell quantity and don't lose its stem cell potential.The second, mescenchymal stem cell technology success in clinical treatment, closely related with the function controlling of mescenchymal stem cell, especially, under the pathology state, mescenchymal stem cell still needs to keep its regenerative power to participate in tissue repair and disease treatment.
The conventional plastics tissue culture system of many employings when mescenchymal stem cell increases in vitro, the amplification in vitro ability of mescenchymal stem cell, repeatedly the differentiation potential after going down to posterity is affected, the high-quality mescenchymal stem cell of sufficient amount not only causes increasing on a large scale fast and effectively at short notice, and make the key function regulation and control of mescenchymal stem cell be affected, can not meet the demand of clinical stem-cell therapy.
Traditional subculture in vitro separately training method expanding stem cells is faced with following predicament and shortcoming:
1), conventional bulk unofficial biography culture mode damages the mescenchymal stem cell self-renewal capacity, makes that the mescenchymal stem cell amplification efficiency reduces, cost is increased.There are some researches show, mesenchymal stem cells MSCs, through after repeatedly going down to posterity, not only can not continue propagation, also loses the ability of its Multidirectional Differentiation simultaneously, and the stem cell in the later stage of going down to posterity generally can only break up to the adipocyte direction.
2), conventional bulk unofficial biography culture mode can not meet the autologous mescenchymal stem cell clinical demand of different ages patient.Current autologous mescenchymal stem cell is applied to clinical treatment gradually, but the stem cell in-vitro multiplication of different ages, directed differentiation ability have very big-difference.There are some researches show, clone's number of the mescenchymal stem cell of vitro culture reduces gradually with the growth at age, by the ratio of itself and bone marrow nucleated cell, is respectively: newborn infant 1:10000; Teenager 1:100000; 50 years old 1:400000; 80 years old 1:1 ~ 2000000.
Large-scale amplification of mesenchymal stem cells in vitro how, when improving its amplification efficiency, and keep the characteristic of its mescenchymal stem cell, is the difficult problem that current mescenchymal stem cell treatment faces.
Summary of the invention
Goal of the invention of the present invention is the deficiency for above-mentioned existing amplification of mesenchymal stem cells technology, and a kind of cell cultures base material and its preparation method and application is provided.The present invention is by umbilical cord fibroblasts to secrete matrix composition cell cultures base material, the cell cultures base material obtained not only has the feature of multiple proteins composition, and can be for the amplification in vitro of mescenchymal stem cell, have advantages of that good biocompatibility, proliferate efficiency are high, stem Cell Phenotypic unanimously, significantly reduces the intracellular reactive oxygen level.
Above-mentioned purpose of the present invention is achieved by following technical solution:
A kind of cell cultures base material, described cell cultures base material comprises that the umbilical cord Fibroblasts in vitro cultivates secreted matrix.
Described umbilical cord inoblast is taken from Huo Tu source, ,Shu source, ,Zhu source, people source.
A kind of preparation method of cell cultures base material, concrete steps are:
S1. obtain the umbilical cord inoblast from Ren Yuan, ,Shu source, pig source Huo Tu source tissue;
S2. configure inducing culture liquid;
S3. cultivate the umbilical cord inoblast and induce umbilical cord fibroblasts to secrete matrix outside ordinary cells is cultivated the base material upper body;
S4. de-cell is processed;
S5. dry, sterilization, described sterilization adopts
60co or ethylene oxide sterilizing.
It is commercial Tissue Culture Dish, culture plate or culturing bottle that the described ordinary cells of step S3 is cultivated base material.
After inducing umbilical cord fibroblasts to secrete matrix concrete steps for the inducing culture liquid that the umbilical cord inoblast is added to step S2 configuration described in step S3, cultivate 7 ~ 20 days, the next day change liquid.
Consisting of of the liquid of inducing culture described in step S2: substratum α-MEM 80 ~ 90% volume ratios, foetal calf serum 10 ~ 20% volume ratios, microbiotic 10 ~ 200 U/mL, VITAMIN B4 0.2 ~ 0.25 mM, stimulating factor component.
Described stimulating factor component is by xitix 10 ~ 100 μ g/mL, proline(Pro) 20 ~ 60 μ g/mL, one or more compositions in dexamethasone 100nM ~ 1 μ M.
Described in step S4, de-cell is treated to first to add and contains NH
4the de-cell treatment solution of OH and Triton X-100 is processed, then adds deoxyribonuclease to process.
Described de-cell treatment solution, by the phosphate buffered saline buffer preparation of pH7.4, contains Triton X-100 1 ~ 5% volume ratio and ammoniacal liquor 0.8 ~ 5% volume ratio.
Described deoxyribonuclease solution, by the phosphate buffered saline buffer preparation of pH7.4, contains 500 ~ 800 U/mL deoxyribonucleases.
The application of cell cultures base material of the present invention in the derived mesenchymal stem cells in vitro amplification.
The application of described cell cultures base material in the derived mesenchymal stem cells in vitro amplification, concrete steps are:
S6. prepare the mescenchymal stem cell nutrient solution, described nutrient solution consists of substratum α-MEM 90% volume ratio, foetal calf serum 10% volume ratio, microbiotic 10 ~ 200 U/mL, VITAMIN B4 0.2 ~ 0.25 mM;
S7. cultivate mescenchymal stem cell, the next day change liquid;
S8. collagenase solution enzymolysis cell, centrifugation.
Collagenase solution described in step S8 is by the phosphate buffered saline buffer preparation of pH7.4, the pancreatin of the collagenase that contains weight ratio 0.1 ~ 1% and weight ratio 0.05%.
Cell cultures base material prepared by the present invention, to utilize umbilical cord inoblast self secretion matrix, after de-cell processing, dry sterilization, the natural tissues engineering materials obtained, contain type i collagen, III Collagen Type VI, fibronectin, ln, decorin etc., can be used for the amplification in vitro of target mescenchymal stem cell.
The natural microenvironment (microtexture, stromatin and the inducible factor etc. that comprise microenvironment in analog) of rebuilding in vitro stem cell growth is to solve increase an on a large scale shortcut of problem of derived mesenchymal stem cells in vitro.In the residing microenvironment of cell, extracellular matrix is being played the part of important role, at the aspects such as attaching, survival, migration, propagation, differentiation and matrix rebuilding of stem cell, is bringing into play keying action.In the microenvironment that MSCs forms in specific extracellular matrix usually, comprising complicated extracellular stress signal and chemical signal, migration, self and the directed differentiation of guiding MSCs.
The extracellular matrix (ECM) that cell cultures base material of the present invention contains the umbilical cord fibroblasts to secrete, not only contain multiple matrix components, comprise I type and III collagen type, fibronectin, Fibronectin, elastin and macromolecular proteoglycan etc., and there is the three-dimensional structure similar to microenvironment in body and snappiness.Simultaneously, the space that after de-cell processing, original cell stays can provide enough growing spaces for later MSCs cultivates (MSCs of cultivation).In three-dimensional ECM microenvironment, inoblast can be synthesized integrin alpha rapidly
5β
1and α
vβ
3contact similar its reaction in vivo with ECM; And, in two-dimentional culture environment or artificial basement membrane, cell needs the long period could produce original adherent behavior.The ECM of emiocytosis can rebuild in vitro with analogue body in microenvironment, regulation and control MSCs behavior, thus affect self-renewal capacity and the directed differentiation potential of MSCs.
Compared with prior art, the present invention has following beneficial effect:
(1), the cell cultures base material for preparing of the present invention, can significantly improve the efficiency that mescenchymal stem cell increases in vitro;
(2), the cell cultures base material for preparing of the present invention, can keep the distinctive immunophenotype of mescenchymal stem cell;
(3), the cell cultures base material for preparing of the present invention, can significantly reduce the active o content of mescenchymal stem cell;
(4), the cell cultures base material for preparing of the present invention has raw material sources characteristics widely, do not produce ethics morals problem;
(5), preparation method of the present invention has that cost is low, method is simple, the characteristics amount of easy handling.
The accompanying drawing explanation
Fig. 1 is the inverted fluorescence microscope figure after the prepared cell cultures base material immunofluorescence dyeing of the present invention;
The cellular form picture that Fig. 2 is the cell cultures base material amplification target mescenchymal stem cell for preparing of the present invention;
Fig. 3 is the increase active oxygen analysis chart of target mescenchymal stem cell of the cell cultures base material that adopts the present invention to prepare and ordinary culture medium material.
Embodiment
Below in conjunction with specific embodiment, the present invention is further explained, but embodiments of the present invention is not limited in any way.Unless stated otherwise, in embodiment, related reagent, method is this area reagent commonly used and method.
embodiment 1
1 utilizes umbilical cord fibroblasts to secrete thing to prepare the cell cultures base material
(1) preparation of inducing culture liquid, its component includes: commercial minimal essential medium α-MEM 80% volume ratio, foetal calf serum 20% volume ratio, microbiotic 10 ~ 200 U/mL, VITAMIN B4 0.25 mM, proline(Pro) 50 μ g/mL, dexamethasone 100nM.
(2) induce umbilical cord fibroblasts to secrete matrix: the density by the umbilical cord inoblast according to 20,000 cell/cm2 is inoculated in commercial Tissue Culture Plate, adds step (3) inducing culture liquid, in 5% the CO of 37oC
2in incubator, cultivate 7 ~ 20 days, the next day change liquid.
(3) de-cell is processed: first with the phosphate buffered saline buffer of pH7.4, clean 3 times, then add the de-cell treatment solution that contains Triton X-100 5% volume ratio and ammoniacal liquor 4% volume ratio, in 5% the CO of 37oC
2in incubator standing 5 minutes, clean 3 times with the phosphate buffered saline buffer of pH7.4, then add and contain 800U/mL deoxyribonuclease solution in 5% the CO of 37oC
2in incubator standing 60 minutes, with the phosphate buffered saline buffer of pH7.4, clean 3 times.
(4) drying: the cell cultures base material after de-cell is processed is carried out to lyophilize.
(5) sterilization: adopt ethylene oxide sterilizing.
The 2 cell cultures base material immunofluorescence dyeings that prepare.
Cell cultures base material progressive type collagen, Collagen Type VI, fibronectin, decorin immunofluorescence dyeing that embodiment 1 is made.The cell cultures base material made for embodiment 1, at first the fixing 15min of ice methyl alcohol, PBS washes 3 times, add 5%BSA sealing 30min, add the primary antibodie with the 1%BSA preparation: Collagen Type VI, Collagen Type VI, fibronectin, decorin are placed in 1h on shaking table, PBS washes 3 times, add coupling FITC bis-anti-, room temperature 30min, PBS washes rear employing inverted fluorescence microscope and takes pictures.As shown in Figure 1, the medium-sized collagen of cell cultures base material, Collagen Type VI, fibronectin enrich expresses result, and content is higher.
embodiment 2
The cellular form of 1 cell cultures base material amplification target mescenchymal stem cell detects
The target mescenchymal stem cell is inoculated in to the cell cultures base material that embodiment 1 obtains, adopts Olympus IX71 microscope fluorescence to take pictures, obtain as Fig. 2 target mescenchymal stem cell form photo.The cell cultures base material is cultivated mescenchymal stem cell and is kept good one-tenth fibre shape, and cell density is high, can be the derived mesenchymal stem cells in vitro amplification foundation is provided.
2
the active oxygen analysis of cell cultures base material prepared by the present invention and ordinary culture medium material amplification target mescenchymal stem cell
The target mescenchymal stem cell is inoculated on the cell cultures base material and common culture plate that embodiment 1 obtains, treats that cell density reaches 90% left and right collecting cell, adopt 2 ', 7 '-dichloro fluorescence acetylacetate (DCFH-DA) detects reactive oxygen species.Cell in 10 μ M DCFH-DA 37 ℃ hatch after 10min hatches, adopt BD dual-wavelength laser flow cytometer to be detected intracellular fluorescence intensity, 10000 cells of each sample collection.As shown in Figure 3, with the ordinary culture medium material, compare, the cell cultures base material that embodiment 1 obtains can obviously reduce active oxygen in the target mescenchymal stem cell, reduces the intracellular reactive oxygen species generation free radical and generates, and effectively the interior environment of Cell protection is stable.
Claims (10)
1. a cell cultures base material, is characterized in that, described cell cultures base material comprises that the umbilical cord Fibroblasts in vitro cultivates secreted matrix.
2. cell cultures base material according to claim 1, is characterized in that, described umbilical cord inoblast is taken from Huo Tu source, ,Shu source, ,Zhu source, people source.
3. the preparation method of the described cell cultures base material of claim 1, is characterized in that, concrete steps are:
S1. obtain the umbilical cord inoblast;
S2. configure inducing culture liquid;
S3. cultivate the umbilical cord inoblast and induce umbilical cord fibroblasts to secrete matrix outside ordinary cells is cultivated the base material upper body;
S4. de-cell is processed;
S5. dry, sterilization.
4. the preparation method of cell cultures base material according to claim 3, is characterized in that, it is commercial Tissue Culture Dish, culture plate or culturing bottle that the described ordinary cells of step S3 is cultivated base material.
5. the preparation method of cell cultures base material according to claim 3, it is characterized in that, after inducing umbilical cord fibroblasts to secrete matrix concrete steps for the inducing culture liquid that the umbilical cord inoblast is added to step S2 configuration described in step S3, cultivate 7 ~ 20 days, the next day change liquid.
6. the preparation method of cell cultures base material according to claim 3, it is characterized in that, consisting of of the liquid of inducing culture described in step S2: substratum α-MEM 80 ~ 90% volume ratios, foetal calf serum 10 ~ 20% volume ratios, microbiotic 10 ~ 200 U/mL, VITAMIN B4 0.2 ~ 0.25 mM, the stimulating factor component.
7. the preparation method of cell cultures base material according to claim 6, is characterized in that, described stimulating factor component is by xitix 10 ~ 100 μ g/mL, proline(Pro) 20 ~ 60 μ g/mL, one or more compositions in dexamethasone 100nM ~ 1 μ M.
8. the preparation method of cell cultures base material according to claim 3, is characterized in that, described in step S4, de-cell is treated to first to add and contains NH
4the de-cell treatment solution of OH and Triton X-100 is processed, then adds deoxyribonuclease to process.
9. the preparation method of cell cultures base material according to claim 8, is characterized in that, described de-cell treatment solution, by the phosphate buffered saline buffer preparation of pH7.4, contains Triton X-100 1 ~ 5% volume ratio and ammoniacal liquor 0.8 ~ 5% volume ratio; Described deoxyribonuclease solution, by the phosphate buffered saline buffer preparation of pH7.4, contains 500 ~ 800 U/mL deoxyribonucleases.
10. the application of cell cultures base material in the derived mesenchymal stem cells in vitro amplification according to claim 1.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103877622A (en) * | 2014-03-26 | 2014-06-25 | 中山大学 | Electrostatic spinning nanofiber-extracellular matrix composite material as well as preparation method and application thereof |
| CN105854084A (en) * | 2016-04-01 | 2016-08-17 | 苏州大学附属第医院 | Extracellular matrix biomaterial for resisting cell premature senility and preparation method and application thereof |
| CN107308497A (en) * | 2017-06-09 | 2017-11-03 | 浙江大学 | The structure of the active microcarrier of nucleus pulposus cell source property |
| CN108295311A (en) * | 2018-03-07 | 2018-07-20 | 南京市第医院 | A kind of preparation method of temperature sensitive type extracellular matrix of kidney hydrogel |
| CN110129274A (en) * | 2019-05-17 | 2019-08-16 | 中国科学院苏州纳米技术与纳米仿生研究所 | Cell matrix materials, preparation method and the application of the cell factor containing gradient |
| CN112538513A (en) * | 2020-12-11 | 2021-03-23 | 湖南美柏生物医药有限公司 | Extracellular matrix MB biological protein and preparation kit and method thereof |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103877622A (en) * | 2014-03-26 | 2014-06-25 | 中山大学 | Electrostatic spinning nanofiber-extracellular matrix composite material as well as preparation method and application thereof |
| CN103877622B (en) * | 2014-03-26 | 2016-04-20 | 中山大学 | A kind of Electrospun nano-fibers-ECM coupled biomaterial and its preparation method and application |
| CN105854084A (en) * | 2016-04-01 | 2016-08-17 | 苏州大学附属第医院 | Extracellular matrix biomaterial for resisting cell premature senility and preparation method and application thereof |
| CN107308497A (en) * | 2017-06-09 | 2017-11-03 | 浙江大学 | The structure of the active microcarrier of nucleus pulposus cell source property |
| CN108295311A (en) * | 2018-03-07 | 2018-07-20 | 南京市第医院 | A kind of preparation method of temperature sensitive type extracellular matrix of kidney hydrogel |
| CN110129274A (en) * | 2019-05-17 | 2019-08-16 | 中国科学院苏州纳米技术与纳米仿生研究所 | Cell matrix materials, preparation method and the application of the cell factor containing gradient |
| CN110129274B (en) * | 2019-05-17 | 2023-06-02 | 中国科学院苏州纳米技术与纳米仿生研究所 | Cell matrix material containing gradient cytokines, preparation method and application thereof |
| CN112538513A (en) * | 2020-12-11 | 2021-03-23 | 湖南美柏生物医药有限公司 | Extracellular matrix MB biological protein and preparation kit and method thereof |
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